CN1102021C - Method for long-term storage of pollen activity - Google Patents

Abstract

A method for storing pollen activity for a long time includes dispersing mature pollen in at least one low-melting-point vegetable oil selected from the group consisting of salad oil, sunflower oil, safflower oil, plant essential oil and vitamin E, freezing at low temperature, taking out the pollen from at least one culture solution selected from the group consisting of C98 culture solution, E98 culture solution and G98 culture solution at 5-80 deg.C, and using oil drop suspension pollen tube method and volatile solvent test method to verify that the pollen in the low-melting-point vegetable oil germinates and survives in a short time.

Description

The method of long term storage pollen activity

The present invention relates to a kind of method of long term storage pollen activity, confirm to realize the pollen tube rudiment at short notice through the pollen of this method long term storage specifically about employing immersion oil freezing method long term storage pollen, and by oil droplet suspension pollen tube rudiment method.

Flower-grower's desire is cultivated flowers in the past, must form seed by the pollen pollination fertilization of flower, gets cultivating seeds again, waits natural factor to influence but the pollen Chang Yin of flower is subjected to weather, field nutrient and soil to contain bacterium (as hay bacillus etc.), and is not easy to be fertilized into seed.Forming the difficult problem of seed for solving the pollen fertilizability, is to have various storage pollen methods to make it not to be subjected to natural factor to influence.At present the laboratory store zooblast or plant gymnocyte the method for normal employing methyl-sulfoxide (DMSO)/freezing process is arranged, but if this method is very big to the injury of pollen when being applied to the storage of pollen, so and be not suitable for storing pollen.If to store bacterium and the used 50v/v% glycerite/freezing method of mould, also be not suitable in addition preserving pollen activity.

In view of this, the inventor forms the cultivation problem of seed for solving the pollen fertilization, avoid pollen influenced by natural factor, it is the method for thinking the long term storage pollen activity, the inventor obtains enlightenment by Butterfly Ginger and lily contain a large amount of essential oils before the natural world environment is not pollinated phenomenon, find to adopt vegetable oil to reach after the immersion oil freezing of low-temperature operation is stored pollen through studying intensively, and make pollen rudiment at short notice with oil droplet suspension pollen tube rudiment method, confirm to adopt the immersion oil freezing can be close to the pollen of surviving indefinite duration, so that finish the present invention.

One of purpose of the present invention is to study the method that a kind of long term storage pollen activity is provided, and promptly immersion oil freezes the method for long term storage pollen, and pollen activity in long term storage can not be subjected to the influence of natural factor and lose.

Two of the object of the invention is to provide a kind of method of long term storage pollen activity, in culture fluid, add the dependency degree of a little electrolyte reduction simultaneously to sugar, solve the problem of generally commonly using agar medium, provide the pollen after immersion oil is freezing can sprout the environment that pipe germinates smoothly.

Three of purpose of the present invention is to provide a kind of method of long term storage pollen activity, and research simultaneously provides the method for the pollen survival condition of a kind of test after immersion oil freezes long term storage, confirms the feasibility of the inventive method.

The method of long term storage pollen activity provided by the invention, be the immersion oil refrigerated storage pollen that adopts vegetable oil and low temperature to extract, also being about to mature pollen is scattered in and is selected from by salad oil, sunflower oil, safflower oil, in at least a low melting point vegetable oil of the colony that plants essential oil and vitamin e form, after sharp freezing, take out, 5～80 ℃ of temperature ranges in being selected from the C98 culture fluid, cultivate at least one culture fluid of the colony of E98 culture fluid and G98 culture fluid and form, make pollen rudiment in the short time in containing a little electrolytical C98 culture fluid after immersion oil is freezing in oil droplet suspension pollen rudiment mode then, confirm to adopt the inventive method can be close to the pollen of surviving indefinite duration, that is pollen is behind the immersion oil freezing storage, taking-up is with oil droplet suspension pollen tube rudiment (drifted droplet pollen germination, hereinafter to be referred as DDPG) under condition, go out the pollen tube rudiment with perusal without microscope short time and cost economy, but confirm that the employing vegetable oil reaches the immersion oil freezing long-term surviving pollen at low-temperature operation.

Above-mentioned vegetable oil is that the inventor is contained a large amount of essential oils as described above and obtained enlightenment under natural environment before ginger and lilium pollen are pollinated, and is by adopting vegetable oil and appropriate solvent to set about with the simulation essential oil, so that finish the present invention.Usual solvents has three classes, and first kind for being usually used in solvent such as DMSO (dimethyl sulfoxide (DMSO)), the glycerine etc. that cell is stored; Second kind is the cleaning agent class, as soft straight chain formula alkyl benzene sulphonate (LinearAlkyl Benzene Sulfonate, LAS), octyl glucoside (OG or octylglucoside), Crematophore etc., wherein OG is the cleaning agent of damaging cells memebrane protein more not; The third is a volatile organic solvent, as ammonia (ammonia), dimethyl amine (dimethyl aminc), phenol (phenol), toluene (toluene), and pyridine acetone (pyridine acetone), butyric acid (butyric acid).Generally being most frequently used in plant with 5%DMSO+10% glucose (glucose) does not have in born of the same parents' the storage of plasmalemma cell, but pollen contacts 5%DMSO at normal temperatures to be mixed with glucose solution behind centrifugal removal solution more again, completely lost the germination survival ability, so and be not suitable for.Though adopt the 50v/v% glycerine water solution not cause pollen impaired immediately again, in-8 ℃ cooling procedure at a slow speed, can cause 98% above pollen death, in-135 ℃ of quenching processes, pollen is all dead again.The inventor adopts and filters out the low melting point vegetable oil for this reason; find material as three different low melting point vegetable oil; for example soybean salad oil, essential oil and synthetic vitamin e oil all manifest the ability (that is the pollen that freezes through long-term oil immersion, still tool is sprouted and managed germinating capacity) of superior protection pollen behind-80 ℃ of freezing storages.

General pollen is sprouted the training bastem and the culture fluid of pipe, Brownbaker and kwack is arranged usually, 1963, the prescription (to call the B/K medium in the following text) that Techniques for Pollination Biologists discloses can adopt, but get bidens through inventor's examination, the Dali chrysanthemum, velvet flowers are observed and adopt above-mentioned traditional B/K medium, remove a little velvet pollen germination (being lower than 20%), all the other are not rudiment all, and with ginger pollen with 22 ℃ of B/K medium 5% Sucrose (sucrose) 1～2 hour, its germination rate only 20%, the inventor finds to be used for pollen by this traditional B/K medium to sprout pipe also not satisfactory.Be that the B/K medium is being contained low high concentration (5%, 20%) adds during sucrose and contain in the culture fluid of 100mg/L potassium nitrate, 280mg/L nitrate of lime, 200mg/L magnesium sulfate, 50mg/L boric acid, agar 1.67%, pipe rudiment situation to pollen is observed, be found in any pollen tube rudiment is not arranged in the culture fluid that is added with 20% sucrose, though and be added with have in the culture fluid of 5% sucrose 1/10 can rudiment but pollen explosion in culture fluid do not grow up, though with 7%, 8% sucrose pollen explosion phenomenon improvement is arranged in addition, but germination rate is not high, still is not suitable for.Sprout the pipe problem for solving this kind pollen, the inventor tries to improve the kind of the sugar in the culture fluid, when adopting sugared concentration to be made as 0.33M,, find when containing sugared B/K medium (6 hours, 22 ℃) different with fructose the best, secondly be lactose, secondly be maltose and trehalose again, relatively poor is sucrose and glucose, galactose, but still fails to make the requirement of the long tube of pollen tube near style physiology length.

Above-mentioned B/K culture fluid is not considered the problem of soda acid buffering, and especially to the cultivation of tiny pollen grain and be not suitable for, because pollen is sprouted the biochemical metabolism effect that pipe relates to certain degree, pollen grain is collected and relied on extraneous column cap, and stable acid or alkali environment is provided.The inventor attempts adding an amount of electrolyte in culture fluid for this reason, phosphoric acid salt for example, and add a small amount of buffer solution and reduce dependence, through attempting taking C98, E98, G98 medium in many ways and adjusting and find to add the optimal medium that becomes that 1.67% agar is made into the culture fluid that adopts 2.83% fructose to sugar.Agar medium commonly used, its used agar is that dehydration reaches gross weight 1/3 at room temperature 2 hours, and the sugar of medium is increased more than 1/2, so agar and sugar must be done suitably to adjust usually, the C98 culture fluid is for containing KNO ₃100 μ g/ml, MgSO ₄200 μ g/ml, Ca (NO ₃) ₂284 μ g/ml, boric acid (Boric acid) 50 μ g/ml, big tomb Taita NO.510 μ g/3ml, fructose (fructose) 4.25g/150ml and agar 2.5g/150ml.

Above-mentioned C98 medium need cooperate immersion oil to freeze to handle, be in this contain the C98 medium through adjusting after in buffer solution and the electrolytical culture fluid beginning can make pollen sprout the pipe germination.In for example single bottle with pollen adding C98 medium, transfer this bottle to high temperature (soaking in the hot water) or ice-cold state, find that freezing merely (for example-10 ℃) cause various pollen dead immediately, also have pollen wall to be even thickening, and pollen wall has the phenomenon of thickening immediately in hot water.Pollen generally at room temperature surpasses again promptly 80% loss of activity (that is in the C98 culture fluid, do not send pollen tube and promptly discharge cellular content, lose the function of normal pollen) a week; Again pollen is left in and also find in the refrigerator that pollen is with loss every days 5～10% and aging gradually.Study intensively through the inventor, find that pollen can stand caloric test rapidly short time (for example 5 minutes in), still can get when returning back to 25 ℃ and normally sprout pipe, the cultivation temperature of pollen is to be advisable at 22～30 ℃.But for solving the problem of pollen long term storage, the inventor finds that its key is appropriate solvent, owing on the flower pesticide of flower thick essential oil is often arranged, pollen is not influenced by external environment and to sprout Guan Yiyu pollen smoothly relevant at the solution of pollen tube at lay up period, how to mix solution similar and medium (liquid) to solution in the pollen tube, make pollen can sprout pipe, that is how to simulate pollen and sprout pipe biochemical metabolism effect, be dealer's desire and break through the problem that solves.Sprout in the factor of pipe at many pollen that influences, the inventor is through meticulously effort, overcomes the difficult problem of medium, freezing, medium respectively, and works out oil droplet suspension pollen tube rudiment method, can be in short-term rudiment survival, so that finish the present invention with confirmation through the pollen of low tempertaure storage.Below describe the inventive method in detail.

The pollen that the inventive method is relevant, can pick up from unifacial leaf and dicotyledonous, wherein unifacial leaf has Liliaceae, Amaryllidaceae, Zingiber, Iridaceae, and dicotyledonous Polemoniaceae etc. is arranged, and the above-mentioned Liliaceae of using for the present invention has lily, flame lily, datlily (tawny daylily), tulip, hyacinth; Amaryllidaceae has Amazon lily, daffodil; Zingiber has butterfly ginger; Iridaceae has sword lily, iris; Polemoniaceae has balloonflower root etc. again.

Be applicable to the vegetable oil of the inventive method; be advisable with the low melting point vegetable oil; wherein with salad oil, sunflower oil, safflower oil, plants essential oil and vitamin e (vitamin e) for more suitable, this all still manifests the ability of superior protection pollen after-80 ℃ of frozen storages at pollen.

Be applicable to the culture fluid of the inventive method, can be the C98 culture fluid, E98 culture fluid and G the culture fluid, (K for example of leading ion in above-mentioned these culture fluids ⁺Ion) is Li ⁺, Na ⁺, Rb ^-Replace or with Sr ⁺⁺Replace the Ca in the culture fluid ⁺⁺Ion, and when keeping same ion strength (Lionic strength) and osmotic pressure, also can obtain same effect (pollen survival).

Above-mentioned culture fluid respectively adopts low concentration sugar in this medium, can make the pollen well-grown.Cultivate the carbohydrate that is suitable in the liquid fructose, lactose, maltose, trehalose, sucrose, glucose, galactose etc. are arranged, be preferably maltose, trehalose and lactose, fructose, be preferably lactose, fructose, the best is a fructose, the concentration of saccharide that is suitable for is 1～10%, is preferable with about 3%.

The agar of using for fixing pollen, be applicable to when being made for medium in the culture fluid of the inventive method, because agar meeting dehydration is more than 1/3, and make the sugar in the culture fluid be condensed into many more than 1/2 than original content, be applicable to the agar in the culture fluid of the inventive method, with 1～3%, be preferably about 1.5～2%, serve as preferred with 1.67%.

The pollen that is applicable to the inventive method is cultivated temperature, can be from 5～80 ℃, and 22～30 ℃ for the most suitable.

In the method for long term storage pollen activity of the present invention, pollen is after adopting low melting point vegetable oil and the immersion oil refrigerated storage with low-temperature operation, still survive for confirming the pollen after the immersion oil refrigerated storage is handled, can sprout the pipe method of testing according to the oil droplet suspension pollen that the inventor researchs and proposes, to confirm pollen rudiment at short notice.

Simultaneously, the present invention also provides a kind of method of testing of storing pollen activity, promptly the pollen of sharp freezing in the low melting point vegetable oil is confirmed rudiment survival in pollen is between short-term in oil droplet suspension pollen tube mode, below explanation oil droplet suspension pollen is sprouted pipe (DDPG) method, and the pollen that the method is cultivated is sprouted Guan Yike and used for cultivating.The step of this method comprises: (1) adds the C98 culture fluid (also can adopt E98, G98 certainly, look pollen and different) of 400～600 μ L in the Clear glass bottles and jars of a 10ml capacity; (2) pollen (take from pollen that immersion oil freezed or from the fresh pollen of flower pesticide) is made it to suspend with a small amount of salad oil (soybean salad oil), sunflower oil, safflower oil, plants essential oil, vitamin e etc., draw above-mentioned suspension 3 μ L and add to the culture fluid liquid level of step (1), the rotating percussion liquid level makes oil droplet be the little floating shape that drips gently; (3) be coated with the vial mouth with vaseline and also cover with little slide, form a closed system, and usually general medium is semi Open System at cultivating process, the material that passes in and out this system is controlled than difficulty.

Is that available inverted phase type microscope is observed at the bottom of directly by bottle or with light-illuminating with the pollen of above-mentioned DDPG method test in (about 1 hour) between short-term, but also naked eyes analysis and observation pollen tube state.

Pollen contains a large amount of essential oils before pollinating under natural environment, inventor's secretion acquisition enlightenment of essential oil thus adopts low melting point vegetable oil (pollen stores and need use the low melting point vegetable oil through freezing to handle, and unlikely destruction cell wall etc. begins) and suitable organic volatile solvent with the simulation essential oil.

Be applicable to that suitable organic solvent of the present invention has: ethanol, acetone, butyric acid, phenol, toluene, arsenic pyridine etc.Pollen is sprouted the enhancement effect of pipe for confirming these organic solvents, the inventor is as the criterion with above-mentioned DDPG method, be that organic solvent is added directly in the DDPG culture fluid, the C98 that adds 3 μ L pollen and 400 μ L～600 μ L in vial (also can add E98 according to circumstances, G98) culture fluid, and get 3 μ L variable concentrations organic volatile solvents respectively to vial, develop the oil droplet suspension pollen that and sprout pipe solvent flashing test (DDPG Solvent Evaporation test, be called for short the DDPGSE test), sprout the pipe rate all more than 95% with the DDPG method with the pollen under above-mentioned this organic solvent of method test.

Be to understand the technical characterictic place of the inventive method, the present invention assesses different culture fluids are sprouted pipe to pollen effect so that to adopt the C98 culture fluid be embodiment and be comparative example with B/K culture fluid (containing 6% sucrose).In addition different pollen are carried out pollen with E98, G98 culture fluid and sprout the pipe test.

Embodiment 1

Get the ginger pollen of fresh maturation and lily pollen through give be dispensed into salad oil and give freeze in-80 ℃ store long-term between after drop into the C98 culture fluid respectively (by KNO ₃100 μ g/ml, MgSO ₄200 μ g/ml, Ca (NO ₃) ₂284 μ g/ml, boric acid 50 μ g/ml, Osuka (big tomb) Taita NO.5 10 μ g/3ml, fructose 4.25g/150ml, agar 2.5g/150ml forms), it is long to measure germination rate (%), average pollen pipe range (mm) and pollen pipe range/pollen body after 25 ℃ of room temperatures are cultivated 60 minutes and 600 minutes down respectively, and it the results are shown in the table 1.

Comparative example 1

Change to the B/K culture fluid (containing 5% sucrose) except that the C98 of embodiment 1 cultivates liquid, all the other are operated with method with embodiment 1, and it the results are shown in table 2.

Table 1

		Cultivated liquid 60 minutes at C98	Cultivated liquid 600 minutes at C98
				Ginger pollen	Germination rate (%)	80％	95％
Pipe range/the pollen body is long for average pollen pipe range (mm) pollen	0.82±0.21mm 10.25	8.4±2.78mm 105
			Lilium pollen		Germination rate (%)	82％	97％
Pipe range/the pollen body is long for average pollen pipe range (mm) pollen	0.73±0.18mm 6.72	12.5±2.50mm 100

Table 2

		Cultivated liquid 60 minutes at B/K	Cultivated liquid 600 minutes at B/K
				Ginger pollen	Germination rate (%)	22％	35％
Pipe range/the pollen body is long for average pollen pipe range (mm) pollen	0.12±0.04mm 1.5	0.36±0.8mm 4.5
			Lilium pollen		Germination rate (%)	15％	25％
Pipe range/the pollen body is long for average pollen pipe range (mm) pollen	0.08±0.03mm 0.64	0.24±0.06mm 1.92

Embodiment 2

Pollen by Iridaceae (as the sword lily iris) is scattered in synthetic vitamin e through giving, and give freeze to handle long-term between after, drop into the G98 culture fluid (by 200 μ L C98-S (C98 sugar-free), 100 μ g NaNO ₃, 100 μ L distilled water, 100 μ L57% sucrose, 100 μ L D-glucoses (35g is in 100ml distilled water) form), at room temperature cultivate the back germination rate and reach 100%.

Embodiment 3

By the pollen of Polemoniaceae (as balloonflower root) through give be scattered in plants essential oil and freezed to handle long-term between after, drop into E98 culture fluid (forming), at room temperature cultivate the back germination rate and reach 100% by 200 μ L C98-S (C-98 sugar-free), 200 μ L distilled water, 200 μ L, 57% sucrose.

Claims (6)

1. A method for long-term storage of pollen activity, characterized in that the mature pollen is dispersed in vegetable oils selected from low-melting point vegetable oils, which include salad oil, sunflower oil, safflower oil, plant essential oils and vitamin E. At least one of the populations, after the pollen is frozen at a low temperature, it is taken out at a temperature range of 5 to 80°C and cultivated in at least one culture medium selected from the group consisting of C98 culture fluid, E98 culture fluid and G98 culture fluid ,in, The C98 culture solution contains potassium nitrate 100 μg/ml, magnesium sulfate 200 μg/ml, calcium nitrate 284 μg/ml, boric acid 50 μg/ml, Otsuka Taita NO.5 10 μg/3ml, fructose 4.25g/150ml and agar 2.5g/ml 150ml; E98 culture solution was prepared from 200 μL sugar-free C98, 200 μL distilled water, and 200 μL 57% sucrose; The G98 culture solution was prepared from 100 μL of dextrose in 100 ml of distilled water from 200 μL of sugar-free C98, 100 μg of sodium nitrate, 100 μL of distilled water, 100 μL of 57% sucrose, and 35 g of glucose in 100 ml of distilled water. 2. The method for long-term storage of pollen activity according to claim 1, wherein the incubation temperature is 22-30°C. 3. The method for long-term storage of pollen activity according to claim 1, characterized in that the concentration of the agar medium in the culture solution is 1-3%; the type of sugar in the culture solution is selected from fructose, lactose, At least one of the group consisting of maltose, trehalose, sucrose, glucose and galactose, the concentration of which is 1-10%, and the main ions in the culture medium are replaced by other ions under the condition of maintaining the same ionic strength and osmotic pressure. replace. 4. The method for long-term storage of pollen activity according to claim 3, characterized in that the concentration of the agar medium in the culture solution is 1.67%, and the sugar in the culture solution is fructose with a concentration of 2.83%. 5. A method for testing the activity of stored pollen, characterized in that the pollen that is frozen in low-melting point vegetable oil is confirmed to germinate and survive in a short period of time by means of oil drop suspended pollen tubes, the steps of the method include: (1) Add 400μL～600μL of a culture solution selected from C98 culture solution, E98 culture solution and G98 culture solution into a 10ml transparent glass bottle; , plant essential oil and vitamin E vegetable oil are suspended, absorb 3 μL of the suspension, and add it to the liquid surface of the culture medium in the previous step (1), gently rotate and hit the liquid surface to make the oil droplets appear as small floating droplets; (3) apply Vaseline Cloth the mouth of a small glass bottle and cover it with a small glass slide to form a closed system for cultivating the germination of pollen for a specified period of time, wherein, The C98 culture solution contains potassium nitrate 100 μg/ml, magnesium sulfate 200 μg/ml, calcium nitrate 284 μg/ml, boric acid 50 μg/ml, Otsuka Taita NO.5 10 μg/3ml, fructose 4.25g/150ml and agar 2.5g/ml 150ml; E98 culture solution was prepared from 200 μL sugar-free C98, 200 μL distilled water, and 200 μL 57% sucrose; The G98 culture solution was prepared from 100 μL of dextrose in 100 ml of distilled water from 200 μL of sugar-free C98, 100 μg of sodium nitrate, 100 μL of distilled water, 100 μL of 57% sucrose, and 35 g of glucose in 100 ml of distilled water. 6. A test method for stored pollen activity according to claim 5, characterized in that the test method also includes directly adding an organic solvent into the culture medium in the form of oil drop suspended pollen germination tubes to promote pollen germination, and putting the pollen in a small glass bottle Add 3 μL of pollen and 400-600 μL of a culture solution selected from C98 culture solution, E98 culture solution and G98 culture solution, and add 3 μL of an organic solvent selected from ethanol, acetone, butyric acid, phenol, toluene and pyridine , operate in the same way as claim 5, to confirm the pollen germination during the specified period of cultivation.